The regulation of cellular growth is mediated in part through the control over the synthesis of the stable RNA species. To study this we have developed an in vitro system active in the transcription, processing and modification of a set of transfer RNAs encoded for by the bacteriophage T4. The goal is to define by fractionation and reconstitution of the system's components, the steps essential to tRNA biosynthesis, and to identify those with regulatory potential. Specific projects include: 1. the transcription in vitro of the tRNAs by the Escherichia coli RNA polymerase, using as templates both T4 DNA and DNA fragments produced by restriction endonucleases; 2. characterization of the initiation step of transcription by study of the conditions and factors influencing it, as well as by analysis of the promoter of transcription by nucleic acid sequencing and cloning of restriction fragments; 3. study of the processing and modification of the tRNAs made in vitro, including characterization of the enzyme, pseudouridine synthetase, which we have purified; 4. studies of the expression of the tRNA genes in vivo, using various techniques, including studies of regulatory mutants, electron microscopy of the transcription apparatas, and radiotracer analysis of tRNA synthesis; 5. preliminary experiments for the development of a similar in vitro system for the transcription of a yeast tRNA gene.